Positive-sense single-stranded RNA (+ssRNA) viruses include highly virulent human pathogens. Within the Flaviviridae family, the genus Flavivirus comprises more than 70 +ssRNA viruses [1], including mosquito-borne Flaviviruses such as Dengue Virus, Yellow Fever Virus, West Nile Virus, Saint Louis Encephalitis Virus, or Japanese Encephalitis Virus and Tick Borne Flaviviruses as Tick-borne encephalitis virus, Omsk Hemorrhagic Fever virus, or Louping ill virus, Flaviviruses with unknown vector like Modoc Virus, Apoi Virus, or Rio Bravo Virus. Dengue virus (DENV), in particular, is an acute viral disease transmitted by mosquito, and one of the most widespread vector-borne viral diseases in humans. Dengue is caused by any of four antigenically distinct serotypes: dengue-1 (DENV1), dengue-2 (DENV2), dengue-3 (DENV3), and dengue-4 (DENV4). There are an estimated 50-100 million cases of dengue fever annually worldwide, half a million of which result in severe forms of the disease, dengue hemorrhagic fever and dengue shock syndrome [2]. Generally, infection with one serotype confers future protective immunity against that particular serotype, but not against the others. In fact, dengue hemorrhagic fever may occur from sequential infection by different virus serotypes in a process called antibody-mediated disease enhancement, where antibodies raised against the first serotype enhance infection with the second serotype [3].
The major envelope glycoprotein (referred to as E protein hereafter) of DENV and of other flaviviruses is responsible for important phenotypic and immunogenic properties of the virion and is believed to lead the virus entry into cells [4,5]. The E protein mediates virus assembly and virus-cell membrane fusion, and initiates infection through binding to cell surfaces. This protein is the principal component of the external surface of the DENV virion and represents the dominant virus antigen, evoking protective immune responses. Dengue serotypes can be distinguished by virus-neutralizing antibodies, but non-neutralizing antibodies against the E protein are cross-reactive. These non-neutralizing antibodies may help bring the virion into close proximity to the normal virus receptor, thus enhancing virus binding and increasing the number of infected cells, with concomitant exacerbation of the disease [6].
While it is well established that the E protein is one of the major proteins responsible for the pathogenicity and immunogenic properties of flaviviruses, the exact residues/regions responsible for these traits remain to be identified. Single-residue substitutions mapped to different parts of the E protein were reported to cause flavivirus attenuation [7], implying that several residues within the E protein are responsible for phenotypic and pathogenic properties. The crystal structure of the soluble ectodomain of DENV2 E protein reveals a hydrophobic pocket lined by residues that influence the pH threshold for membrane fusion [8-10]. The protein has three structural domains (DI, DII, DIII) that map closely to the three antigenic regions (C, A, and B, respectively) [4]. DENV enters the host cell when the E protein binds to a yet undefined cell receptor and responds to a reduced pH of the endosome by a conformational change [1,1]. This conformational change induces fusion of the virus and the host cell membrane. The crystal structure of DENV3 E protein indicates that the serotype-specific mutations that allow viral evasion from immune surveillance (neutralization escape mutations) are all located on the surface of domain III, which has been implicated in receptor binding [12, 13]. The apparent involvement of the host immune system in disease pathogenesis, the so-called antibody-dependent enhancement (ADE), has hampered development of a vaccine against dengue. Therefore it is important to identify specific regions of the E protein having a high potential success as targets in order to develop robust diagnostics and vaccines against Flavivirus, specifically dengue virus.